Azadirachta indica A. Juss., Meliaceae, is a medicinal plant known as Indian neem or lilac, Persian lilac or margosa tree, originated from Asia, but worldwide cultivated. In Brazil, neem or nim is well adapted in the northeast Caatinga biome being largely recognized by its repellent and insecticide properties causing no environment pollution (Martinez, 2002). In folk medicine, oil seeds, bark and leaf extracts have been used to treat gastrointestinal, analgesic and inûammatory disorders. Experimental data demonstrated immunomodulator and anti-inflammatory activities of bark and leave extracts and also antipyretic and anti-inflammatory effects of oil seeds (Arora et al., 2011; Biswas et al., 2002).

Many compounds have been isolated from the oil seeds (mixture of tetranortripterpenes) and barks (polysaccharides) of A. indica. Although studies have described anti-inflammatory effect of polysaccharide extracts (Biswas et al., 2002), little has identified the bioactive components. Based on previous knowledge of A. indica constituents and their occurrence in traditional medicinal preparations, this study evaluated the effects of isolated polysaccharide fractions of A. indica seed tegument in models of inflammation in vivo.

Ten milligrams of TPL, dissolved in 20 mL of H20 (1:2, w/v), was applied to ion exchange chromatography (DEAE-cellulose), equilibrated and washed with distilled water for removal of neutral polysaccharides, being the acidic polysaccharides eluted (60 mL/h) with NaCl (0.25-1 M). TPL and major polysaccharide fractions were dialyzed and further lyophilized. Content of carbohydrate (Dubois et al., 1956), uronic acid (Dische, 1947) and protein (Bradford et al., 1976) were assessed using D-galactose, D-galacturonic acid and BSA as respective standards.

Animals

Wistar rats (150-200 g) were maintained at 25 ºC, under a 12/12 h light/dark cycle, with free access of food and water. Protocols were approved by the Institutional Animal Care and Use Committee of the State University of Ceará-UECE (09204632-0) in accordance with the Guide for the Care and Use of Laboratory Animals of the US Department of Health and Human Services (NIH publication n° 85-23, revised 1985).

As showed in the paw edema model, FI effectively inhibited the effect of late-phase inflammatory mediators involved in cell events. In fact, FI (0.1 mg/kg) reduced the number of total leukocyte migration in 67% when stimulated with fMLP (2.74±0.15; FI 0.89±0.13 x 103) and in 48% when stimulated with carrageenan (7.79±0.03; FI 4.07±0.04 x 103) (Figure 2E). Neutrophils were the most reduced cells, about 75%, when stimulated with fMLP (1.50±0.20; FI 0.37±0.02 x 103) and about 55% when stimulated with carrageenan (5.01±0.03; FI 4.07±0.04 x 103) (Figure 2E). It is well known that in acute inflammatory responses the neutrophil rolling/ adhesion along endothelium is a result of direct (fMLP) or indirect activation (carrageenan), via resident cells, that culminate in chemotactic release of mediators (Ribeiro et al., 1997). Thus, FI may be interfering in both pathways. Accordingly, pectic polysaccharide fractions from aerial parts of Comarum palustre (Popov et al., 2005) and the acidic polysaccharide from flower-heads of Cyrtopodium cardiochilum (Barreto & Parente, 2006) were shown to inhibit cell migration. Further, FI (±0.03 x 103) reduced vascular leakage elicited by carrageenan (0.9±0.06 x 103 mg/mL) in 81% (Figure 2F), corroborating the initial phase action of FI in the model of paw edema. This finding stretches the role of FI in inflammatory vascular events, as described for polysaccharide fractions of Orbignya phalerata mesocarp (Pereira da Silva & Paz Parente, 2001).

Moreover, FI was well tolerated by the animals, since the observed lack of lethality or other external symptoms. However, minor alterations were seen in the wet weigh (g) of stomach (1.53±0.05; control 1.76±0.16) and kidney (0.93±0.03; control 0.80±0.03). Alanine and aspartate transaminases (ALT; AST), creatinine, albumin and globulin levels were not affected, but urea levels increased. The number of circulating lymphocytes were also raised (Table 2), which could be accredited in part to the immunomodulatory action of plant polysaccharides (Arora et al., 2011).

In conclusion, TPL and FI fractioned from A. indica seed tegument exhibited potent anti-inflammatory activity, interfering not only in vascular, but especially in cellular inflammatory events, involving serotonin, PGE2 and NO. These polysaccharides could be revealed as important active components in traditionally prepared remedies to treat inflammatory diseases.

Acknowledgements

Authors are grateful to Ms. Vaneicia dos S. Gomes, who identified the plant species and Gabriela F. O. Marques-Domingos for laboratory technical support. This research was funding by Brazilian grants from CNPq, CAPES and FUNCAP. AMS Assreuy is senior investigator of CNPq.